Rapid Detection of Known Mutations and Its Application to Carrie Testing

本研究主要进行了以下几个方面的工作：1.全羧化酶合成酶（HCS）基因的克隆缺乏HCS可引起生物素反应性多羧化酶。我们已经克隆了人HCSCDNA，其显示与BirA的同源性并定位于染色体21q22.1.2。各种遗传性疾病的突变研究：1）6例日本患者的突变分析显示T997C，G1935A和一个碱基缺失（G1067）的错义突变。这些突变共同占日本HCS等位基因的约83%。2)日本GSD患者中R83 H、P257 L、R170 X、g727 t和IVS 1nt-a共占98%，其中g727 t剪接突变最常见。3)PKU患者的R413P、IVS-4nt-1、R111X、Y204C、R243Q、R252W、R241C、R278I和IVS-9突变占日本PKU突变等位基因的67%。4)PKU患者的R243Q、R413P、IVS-4、R365X、R111X、R261Q、Y204C突变占中国人PKU突变等位基因的61%。5)日本甲基丙二酸血症的突变; G425 T和769 Δ。6）日本CPT II缺陷患者的突变; F352 C、V368 I和M647 V。7）日本家族性ALS的突变; H46 R。多重等位基因特异性PCR方法的建立我们应用ASPCR方法检测PAH基因突变。在日本和中国发现的12种突变已开发出单一ASPCR检测。3或4个突变的ASPCR反应已经被多重化。两种多重检测将分别检测R111X、R261Q和Y204C突变以及R243Q、R413P、IVS 4nt-1和Y356X突变的存在。

Present study carried out the following projects ;1. Cloning of holocarboxylase synthetase (HCS) geneDeficiency of HCS causes biotin-responsive multiple carboxylase. We have choned the human HCScDNA,which shows homology to BirA and maps to chromosome 21q22.1.2. Mutation studies from various genetic disorders :1) Mutation analysis in 6 Japanese patients revealed the misssense mutation of T997C,G1935A and one base deletion (G1067). These mutations together account for about 83% of Japanese HCS alleles. 2) Mutations of GSD patients ; R83H,P257L,R170X,g727t and IVS 1nt-a, together account for 98% of Japanese GSD mutant alleles.The g727t splicing mutation was the most common mutation among the Japanese patients. 3) Mutaions of PKU patients ; R413P,IVS-4nt-1, R111X,Y204C,R243Q,R252W,R241C,R278I and IVS-9, account for 67% of Japanese PKU mutant alleles. 4) Mutaions of PKU patients ; R243Q,R413P,IVS-4, R365X,R111X,R261Q,Y204C,account for 61% of Chinese PKU mutant alleles. 5) Mutations of Japanese methylmalonic acidemia ; G425T and 769DELTACA.6) Mutations of Japanese CPT II deficient Patients ; F352C,V368I and M647V.7) Mutation of Japanese familial ALS ; H46R.3. Development of multiplex allele specific PCR ; We have applied ASPCR methodology to the detection of mutations in PAH gene. Single ASPCR tests have been developed for 12 mutations found in Japan and China. ASPCR reactions for 3 or 4 mutaions have been multiplexed. The two multiplex tests will detect the presence of the R111X,R261Q and Y204C mutations and the R243Q,R413P,IVS 4nt-1 and Y356X mutations, respectively.